白山毛桃根提取物对非小细胞肺癌细胞的抑制效果及机制研究

Inhibitory effect of extracts of radix actnidiae erianthae on non-small cell lung cancer cells by regulating miR-182-5p/PCDH10 expression

目的:探讨白山毛桃根提取物对非小细胞肺癌细胞增殖、迁移和侵袭的影响,并探究其潜在分子调控机制。方法:设置白山毛桃根提取物各浓度(0 mg/mL、10 mg/mL、20 mg/mL、40 mg/mL、60 mg/mL)实验组进行MTT实验,观察白山毛桃根提取物对肺癌H1299细胞增殖的影响。根据MTT实验结果,再设置分组为对照组(无白山毛桃根提取物)、白山毛桃根提取物组(浓度40 mg/mL)、白山毛桃根提取物+miR-NC组、白山毛桃根提取物+miR-182-5p组进行实验。采用脂质体法,分别将miRNC、miR-182-5pmimics、anti-miR-NC、anti-miR-182-5p转染至H1299细胞。Transwell检测各组细胞的侵袭和迁移;实时荧光定量PCR(qPCR)检测H1299细胞中miR-182-5pmRNA和PCDH10 mRNA表达水平;蛋白免疫印迹(Western blotting)检测PCDH10蛋白表达;双荧光素酶报告基因检测实验检测荧光活性。结果:各浓度白山毛桃根提取物组细胞抑制率均高于0 mg/mL组,并呈剂量依赖性(P<0.05)。荧光素酶报告基因实验结果显示,miR-182-5p组WT-PCDH10非小细胞肺癌细胞的荧光素酶活性低于miR-NC组(P<0.05);而miR-182-5p组与miR-NC组的MUT-PCDH10荧光素酶活性比较,差异无统计学意义(P>0.05),白山毛桃根提取物组细胞迁移和侵袭数量低于对照组(P<0.05);白山毛桃根提取物组H1299细胞中miR-182-5p mRNA表达低于对照组;PCDH10 mRNA及蛋白表达高于对照组(P<0.05),白山毛桃根提取物组H1299细胞中miR-182-5p表达水平、细胞迁移和侵袭数量低于对照组;白山毛桃根提取物+miR-24-3p组H1299细胞中miR-182-5p表达水平、细胞迁移和侵袭数量高于白山毛桃根提取物+miR-NC组(P<0.05);白山毛桃根提取物组H1299细胞中PCDH10蛋白表达水平、细胞抑制率高于对照组;白山毛桃根提取物+miR-182-5p组H1299细胞中PCDH10蛋白表达水平、细胞抑制率低于白山毛桃根提取物+miR-NC组(P<0.05)。结论:白山毛桃根提取物可抑制非小细胞肺癌H1299细胞增殖、迁移和侵袭,其机制可能与miR-182-5p调控PCDH10基因表达有关。

Objective:To explore the effects of extractive of radix actnidiae erianthae on the proliferation and metastasis of non-small cell lung cancer cells and its potential mechanism.Methods:Firstly,the experimental groups were set up with different concentrations(0,10,20,40,60 mg/ml)of the radix actnidiae erianthae.The MTT test was carried out to observe the effect of the extract on the proliferation of lung cancer H1299 cells.According to the results of MTT experiment,the experiment was further divided into control group(no extractive of radix actnidiae erianthae),extractive of radix actnidiae erianthae group(concentration 40 mg/ml),extractive of radix actnidiae erianthae+miR-NC group,extractive of radix actnidiae erianthae+miR-182-5pgroup.The miR-NC,miR-182-5p mimics,anti-miR-NC and anti-miR-182-5p were transfected into H1299 cells by liposome method,respectively.MTT assay was used to detect cell proliferation in each group.Transwell was used to detect the invasion and migration of cells in each group;qRT-PCR was used to detect the expression of miR-182-5pand PCDH10 mRNAin non-small cell lung cancer H1299 cells;Western blot was used to detect PCDH10 protein expression;Dual luciferase reporter gene assay was used to detect fluorescence activity.Results:Compared with the 0 mg/ml group,the inhibition rate of cells in different concentrations of extractive of radix actnidiae erianthae group was significantly increased(P<0.05).Compared with the control group,the migration and invasion of cells in the extractive of radix actnidiae erianthae group were significantly decreased(P<0.05);the expression levels of the miR-182-5p was significantly decreased,expression levels of PCDH10 mRNA and protein were significantly increased(P<0.05).Compared with the anti-miR-NC group,the number of migration and invasion of H1299 cells in the anti-miR-182-5p group was significantly decreased(P<0.05).Conclusion:Extractive of radix actnidiae erianthae inhibits proliferation,migration and invasion of non-small cell lung cancer H1299 cells.Overexpression of miR-182-5p reversed the inhibitory effect of extractive of radix actnidiae erianthae on proliferation,migration and invasion of H1299 cells,and its mechanism may be related to the regulation of miR-182-5ponPCDH10 gene expression.